Material collection and sample preparation
Bamboo shoots were collected from eight locations (Fig. 1), six of which were located in Yamagata Prefecture. Four were collected from the Shonai region, which is the main production area in the prefecture, one in Yuza Town, one in Mikawa Town, and two in Tsuruoka City (Sanze and Haguro), while the other two were collected from the inland region, one in Yamanobe Town and one in Asahi Town. To compare the results with those of other prefectures, one site in Kyoto Prefecture and one site in Fukuoka Prefecture were selected from the top production regions, which are south of western Japan [4].
Three bamboo shoots were collected from each site. Because the developmental time for bamboo shoots varies from region to region, the collection period was approximately 10 days after the first bamboo shoot was confirmed at each site, which ranged from early to late May in 2017. The size of the bamboo shoots collected was standardized to the Shonai area’s standard M (600–1000 g) for fresh food shipments [5]. The bamboo shoots were collected from the two locations outside of the prefecture from early to mid-April in 2018, and three bamboo shoots that met the same raw food shipping standard M (600–1000 g) used for those collected within the prefecture were selected.
Fresh edible bamboo shoots collected in the prefecture are distributed mainly through in-prefecture market shipments and direct sales, with the shortest time from collection to sale being approximately one day, and they are rarely kept refrigerated during market management or sales [5]. Therefore, assuming similar distribution conditions, bamboo shoots were transported to the Yamagata Prefecture Forestry Research and Training Center within 3 h of collection, stored in a well-ventilated area out of direct sunlight until 24 h after collection, and then fixed by freezing. Specimens collected in Kyoto and Fukuoka prefectures were also transported and stored under conditions similar to those in Yamagata Prefecture, and they were transported by refrigerated vehicle to the Yamagata Prefecture Forest Research and Training Center within 2 days. Upon arrival, the specimens were immediately frozen and fixed.
All the frozen bamboo shoots were freeze-dried for approximately 3 days and used as samples. The freeze-drying does not greatly alter the processing methods, and the component levels before drying are mostly maintained [20,21,22]. Because there is no change in flavor that would cause the food to lose its original characteristics, there is no effect on sensory testing or taste sensor and chemical analyses [20].
Analysis of the “egumi” taste of bamboo shoots by region.
Chemical analysis
Homogentisic acid and oxalic acid, which are considered to be the main components of “egumi” taste, were analyzed in one sample from Shonai area in Yamagata Prefecture (Yuza Town), one sample from inland area in Yamagata Prefecture (Asahi Town), and one sample from outside the prefecture (Fukuoka Prefecture).
Homogentisic acid was analyzed using gas chromatograph–mass spectrometer (GC–MS). Each of the freeze-dried samples was degreased, and they were extracted three times by immersion in methanol to recover the supernatant. After concentrating the recovered supernatant, it was transformed into an extract in a constant volume of methanol. The solvent was removed under vacuum conditions to obtain a methanol extract. The methanol extract was analyzed by GC–MS after trimethylsilyl derivatization by TMSI-H [hexamethyldisilazane and trimethylchlorosilane in pyridine (2:1:10, v/v)] (GL Science Inc., Tokyo, Japan) and N,O-bis(trimethylsilyl) acetamide (GL Science Inc., Tokyo, Japan). The GC–MS data were collected with a Shimadzu QP-2010 Ultra-high performance gas chromatograph–mass spectrometer (Shimadzu, Kyoto, Japan) under the following conditions: SH-Rtx-5ms capillary column [30 m × 0.25 mm inner diameter; 0.25-μm film thickness (Shimadzu, Kyoto, Japan)], column temperature ranging from 100 °C (1 min) to 320 °C (5 min) at 5 °C/min, injection temperature of 250 °C, interface temperature of 320 °C, acquisition mass range of 50–800 atomic mass units, and helium as the carrier gas.
The concentration of oxalic acid was measured using an E-kit oxalic acid manufactured by J.K. International Co. Measurements were performed on the three selected samples as in GC–MS. 20 g (fresh weight) of each freeze-dried sample was weighed, and distilled water was added to form a 20-fold dilution. The treatment time for the test was 6 h, and the samples were refrigerated to avoid deterioration owing to temperature. Afterwards, the samples were thoroughly agitated with a mixer and centrifuged at 3000 rpm for 5 min, and the collected supernatants were then used. The supernatants were kept frozen until just before the analyses. The treatment method, spectrophotometry conditions, and formula for calculating the oxalic acid concentration were described in the manual provided with the kit.
Taste analysis
The samples used were tenfold diluted solutions prepared by the same method as in oxalic acid concentration measurement test.
Two types of taste sensors, αASTREE (Alpha M.O.S. Japan K.K., Tokyo, Japan) and TS-5000Z (Intelligent Sensor Technology, Inc., Kanagawa, Japan), were used for the analysis.
αASTREE classifies flavors by combining seven types of sensors that exhibit different response spectra to create a unique pattern for each measurement sample [23]. Each sensor consists of a chemical field effect transistor (CHEMFET) base coated with a different copolymer as a sensitive film [23, 24]. In general, most CHEMFET sensors used in industry and research are designed with membranes that respond only to certain ionic species, but the sensors used in αASTREE have been developed with sensitive materials to induce interactions (such as hydrogen bonds and van der Waals interactions) with a wide range of dissolved taste components, and are sensitive to many ionic and neutral species in complex taste solutions [23, 24]. The potential difference between the CHEMFET sensor and the reference electrode (Ag/AgCl) changes when the sensing membrane comes into contact with the taste component in the sample solution, and is detected as a signal [23, 24]. In αASTREE, three tastes (sour, salty, and umami) are analyzed directly from the corresponding sensor output, while tastes with other attributes (astringent, bitter, pungent, etc.) need to be defined for each analysis. Analyses using the standard addition method are recommended [23]. In the standard addition method, influential raw materials, and reference materials that are considered to influence the taste to be evaluated, are selected and at least two concentrations are added. In this study, oxalic acid, which was confirmed to be contained in the chemical analysis described above, was used as the target of the standard addition method. Three concentrations of oxalic acid solutions (1 mM, 10 mM, and 100 mM) were used for taste sensor analysis, and principal component analysis was performed along with the analysis results of the samples. In the taste sensor analysis, each sample was measured six times, the average of the two measurements in the second half, when the data was judged to be stable, was used for the principal component analysis. All the principal component analyses were performed using Bell Curve for Excel version 3.21 (Social Survey Research Information Co., Ltd.).
The TS-5000Z employs an artificial lipid membrane taste sensor that mimics the taste recognition mechanism of living organisms [14]. The membrane potential change generated by electrostatic or hydrophobic interactions with the taste substance is detected by the computer as the sensor output, and the potential difference between each sample [25]. The output from each sensor is converted into the corresponding specific “taste item” [25]. The TS-5000Z uses the CPA measurement method, which detects two types of information from a single sensor [25]. Specifically, the potential of the reference solution is set to zero, and the potential difference between the reference solution and the sample solution is measured as a pre-taste [25]. After that, the sensor is lightly cleaned, and the potential difference when the reference solution is measured again is measured as the aftertaste [25].
However, TS-5000Z does not have a sensor that directly measures “egumi” taste, which is not a basic taste. In addition, there is no clear definition of “egumi” taste; it is reported to be a complex mixture of bitter and astringent tastes [26], or a mixture of bitter and astringent tastes with an itchy sensation [27]. For this reason, the measurements of bitterness (first taste), general bitterness (aftertaste), astringent stimulation (first taste), and astringent taste (aftertaste) were included in the analysis in this study. Measurements were made using the three selected samples as in chemical analysis.